Candida tropicalis cells and use thereof

ABSTRACT

The invention relates to genetically engineered  Candida tropicalis  cells, use thereof and a method of production of ω-hydroxycarboxylic acids and ω-hydroxycarboxylic acid esters.

FIELD OF THE INVENTION

The invention relates to genetically engineered Candida tropicaliscells, use thereof and a method of production of ω-hydroxycarboxylicacids and ω-hydroxycarboxylic acid esters.

BACKGROUND OF THE INVENTION

Owing to its ability to form dicarboxylic acids from alkanes, Candidatropicalis is a well-characterized ascomycete.

WO91/006660 describes Candida tropicalis cells that are completelyinhibited in β-oxidation through interruption of the POX4 and/or POX5genes, and achieve increased yields of α,ω-dicarboxylic acids.

WO00/020566 describes cytochrome P450 monooxygenases and NADPHcytochrome P450 oxidoreductases from Candida tropicalis and use thereoffor influencing ω-hydroxylation, the first step in ω-oxidation.

WO03/089610 describes enzymes from Candida tropicalis which catalyse thesecond step of ω-oxidation, the conversion of a fatty alcohol to analdehyde, and use thereof for improved production of dicarboxylic acids.

The cells and methods described so far are not suitable for theproduction of ω-hydroxycarboxylic acids or their esters, as theω-hydroxycarboxylic acids are always only present as a short-livedintermediate and are immediately metabolized further.

ω-Hydroxycarboxylic acids and their esters are economically importantcompounds as precursors of polymers, and this forms the basis of thecommercial usability of the present invention.

The task of the invention was to find a way of preparingω-hydroxycarboxylic acids or ω-hydroxycarboxylic acid esters byfermentation in sufficient amounts, in particular in the mediumsurrounding the cells.

DESCRIPTION OF THE INVENTION

It was found, surprisingly, that the cells described hereunder make acontribution to solution of this task.

The object of the present invention is therefore a cell as described inclaim 1.

Another object of the invention is the use of the cell according to theinvention and a method of production of ω-hydroxycarboxylic acids andω-hydroxycarboxylic acid esters using the cells according to theinvention.

Advantages of the invention are the gentle conversion of the educt usedto the ω-hydroxycarboxylic acids and corresponding esters and a highspecificity of the method and an associated high yield based on theeduct used.

One object of the present invention is a Candida tropicalis cell, inparticular one from the strain ATCC 20336, which is characterized inthat the cell has, compared with its wild type, a reduced activity of atleast one of the enzymes that are encoded by the intron-free nucleicacid sequences selected from the two group comprising

A) Seq ID No. 1, Seq ID No. 3, Seq ID No. 5, Seq ID No. 7, Seq ID No. 9,Seq ID No. 11, Seq ID No. 13, Seq ID No. 15, Seq ID No. 17, Seq ID No.19, Seq ID No. 21, Seq ID No. 23, Seq ID No. 25, Seq ID No. 27, Seq IDNo. 29, Seq ID No. 31, Seq ID No. 33, Seq ID No. 35, Seq ID No. 37, SeqID No. 39, Seq ID No. 41, Seq ID No. 43, Seq ID No. 45, Seq ID No. 47,Seq ID No. 49, Seq ID No. 51, Seq ID No. 53, Seq ID No. 55, Seq ID No.57, Seq ID No. 59, Seq ID No. 61, Seq ID No. 63, Seq ID No. 65 and SeqID No. 67; in particular Seq ID No. 1, Seq ID No. 3, Seq ID No. 5, SeqID No. 7, Seq ID No. 9, Seq ID No. 11, Seq ID No. 13, Seq ID No. 15, SeqID No. 17, Seq ID No. 19, Seq ID No. 21, Seq ID No. 23, Seq ID No. 25,Seq ID No. 27, Seq ID No. 29, Seq ID No. 31, Seq ID No. 33, Seq ID No.35, Seq ID No. 37, Seq ID No. 39, Seq ID No. 41, Seq ID No. 43, Seq IDNo. 45, Seq ID No. 47, Seq ID No. 49 and Seq ID No. 51; quite especiallySeq ID No. 1, Seq ID No. 3, Seq ID No. 5, Seq ID No. 7, Seq ID No. 9,Seq ID No. 11, Seq ID No. 13, Seq ID No. 15, Seq ID No. 17, Seq ID No.19, Seq ID No. 21, Seq ID No. 23, Seq ID No. 25 and Seq ID No. 27,B) a sequence that is identical to at least 80%, especially preferablyto at least 90%, even more preferably to at least 95% and mostpreferably to at least 99% to one of the sequences Seq ID No. 1, Seq IDNo. 3, Seq ID No. 5, Seq ID No. 7, Seq ID No. 9, Seq ID No. 11, Seq IDNo. 13, Seq ID No. 15, Seq ID No. 17, Seq ID No. 19, Seq ID No. 21, SeqID No. 23, Seq ID No. 25, Seq ID No. 27, Seq ID No. 29, Seq ID No. 31,Seq ID No. 33, Seq ID No. 35, Seq ID No. 37, Seq ID No. 39, Seq ID No.41, Seq ID No. 43, Seq ID No. 45, Seq ID No. 47, Seq ID No. 49, Seq IDNo. 51, Seq ID No. 53, Seq ID No. 55, Seq ID No. 57, Seq ID No. 59, SeqID No. 61, Seq ID No. 63, Seq ID No. 65 and Seq ID No. 67; in particularto Seq ID No. 1, Seq ID No. 3, Seq ID No. 5, Seq ID No. 7, Seq ID No. 9,Seq ID No. 11, Seq ID No. 13, Seq ID No. 15, Seq ID No. 17, Seq ID No.19, Seq ID No. 21, Seq ID No. 23, Seq ID No. 25, Seq ID No. 27, Seq IDNo. 29, Seq ID No. 31, Seq ID No. 33, Seq ID No. 35, Seq ID No. 37, SeqID No. 39, Seq ID No. 41, Seq ID No. 43, Seq ID No. 45, Seq ID No. 47,Seq ID No. 49 and Seq ID No. 51; quite especially to Seq ID No. 1, SeqID No. 3, Seq ID No. 5, Seq ID No. 7, Seq ID No. 9, Seq ID No. 11, SeqID No. 13, Seq ID No. 15, Seq ID No. 17, Seq ID No. 19, Seq ID No. 21,Seq ID No. 23, Seq ID No. 25 and Seq ID No. 27.

In this connection, the nucleic acid sequence group that is preferredaccording to the invention is group A).

A “wild type” of a cell preferably means, in connection with the presentinvention, the starting strain from which the cell according to theinvention was derived by manipulation of the elements (for example thegenes comprising the aforesaid nucleic acid sequences coding for acorresponding enzyme or the promoters contained in the correspondinggene, which are linked functionally with the aforesaid nucleic acidsequences), which influence the activities of the enzymes encoded by thestated nucleic acid Seq ID No. If for example the activity of the enzymeencoded by Seq ID No. 1 in the strain ATCC 20336 is reduced byinterruption of the corresponding gene, then the strain ATCC 20336 thatis unchanged and was used for the corresponding manipulation is to beregarded as the “wild type”.

The term “gene” means, in connection with the present invention, notonly the encoding DNA region or that transcribed to mRNA, the“structural gene”, but in addition promoter, possible intron, enhancerand other regulatory sequence, and terminator, regions.

The term “activity of an enzyme” always means, in connection with theinvention, the enzymatic activity that catalyses the reactions of12-hydroxydodecanoic acid to 1,12-dodecane diacid by the entire cell.This activity is preferably determined by the following method:

Starting from a single colony, a 100-ml Erlenmeyer flask with 10 ml ofYM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone and 1.0%(w/w) glucose) is cultivated at 30° C. and 90 rpm for 24 h. Then,starting from this culture, 10 ml is inoculated into a 1-litreErlenmeyer flask with 100 ml of production medium (for 1 litre: 25 gglucose, 7.6 g NH₄Cl, 1.5 g Na₂SO₄, 300 ml of a 1 mM potassium phosphatebuffer (pH 7.0), 20 mg ZnSO₄×7H₂O, 20 mg MnSO₄×4H₂O, 20 mg nicotinicacid, 20 mg pyridoxine, 8 mg thiamine and 6 mg pantothenate). It iscultivated for 24 h at 30° C.

After 24 h, 12-hydroxydodecanoic acid is added to the cell suspension,so that the concentration is not greater than 0.5 g/l. Glucose orglycerol is also added as co-substrate, so that the concentration of theco-substrate does not drop below 0.2 g/l. After 0 h, 0.5 h, 1 h, andthen hourly up to a cultivation time of 24 h, samples (1 ml) are takenfor measurement of 12-hydroxydodecanoic acid, 12-oxo-dodecanoic acid and1,12-dodecane diacid and the corresponding methyl esters, and forchecking the cell count. After each measurement, the pH is kept between5.0 and 6.5 with 6N NaOH or 4NH₂SO₄. During cultivation, cell growth isverified by checking the “colony forming units” (CFU). The decrease of12-hydroxydodecanoic acid and the production of 1,12-dodecane diacid orthe corresponding methyl esters are verified by LC-MS. For this, 500 μlof culture broth is adjusted to pH 1 and then extracted with the samevolume of diethyl ether or ethyl acetate and analysed by LC-MS.

The measuring system consists of an HP1100 HPLC (Agilent Technologies,Waldbronn, Germany) with degasser, autosampler and column furnace,coupled to a mass-selective quadrupole detector MSD (AgilentTechnologies, Waldbronn, Germany). Chromatographic separation isachieved on a reversed phase e.g. 125×2 mm Luna C18(2) column(Phenomenex, Aschaffenburg, Germany) at 40° C. Gradient elution isperformed at a flow of 0.3 ml/min (A: 0.02% formic acid in water and B:0.02% formic acid in acetonitrile). Alternatively, the organic extractsare analysed by GC-FID (Perkin Elmer, Rodgau-Jügesheim, Germany).Chromatographic separation is performed on a methylpolysiloxane (5%phenyl) phase e.g. Elite 5, 30 m, 0.25 mm ID, 0.25 μm FD (Perkin Elmer,Rodgau-Jügesheim, Germany). Before measurement, a methylation reagente.g. trimethylsulphonium hydroxide “TMSH” (Macherey-Nagel GmbH & Co. KG,Düren, Germany) is added to free acids and on injection they areconverted to the corresponding methyl esters.

By calculating the measured concentration of 1,12-dodecane diacid andthe cell number at the time of sampling, it is possible to determine thespecific production rate of 1,12-dodecane diacid from12-hydroxydodecanoic acid and therefore the “activity of an enzyme” in acell as defined above. The formulation “reduced activity compared withits wild type” means an activity relative to the wild-type activitypreferably reduced by at least 50%, especially preferably by at least90%, more preferably by at least 99.9%, even more preferably by at least99.99% and most preferably by at least 99.999%.

The decrease in activity of the cell according to the invention comparedwith its wild type is determined by the method described above fordetermining activity using cell numbers/concentrations as identical aspossible, the cells having been grown under the same conditions, forexample medium, gassing, agitation.

“Nucleotide identity” relative to the stated sequences can be determinedusing known methods. Generally, special computer programs are used withalgorithms taking special requirements into account. Preferred methodsfor determining identity first produce the greatest agreement betweenthe sequences to be compared. Computer programs for determining identitycomprise, but are not restricted to, the GCG software package, including

-   -   GAP (Deveroy, J. et al., Nucleic Acid Research 12 (1984), page        387, Genetics Computer Group University of Wisconsin, Medicine        (Wi), and    -   BLASTP, BLASTN and FASTA (Altschul, S. et al., Journal of        Molecular Biology 215 (1990), pages 403-410. The BLAST program        can be obtained from the National Center for Biotechnology        Information (NCBI) and from other sources (BLAST Manual,        Altschul S. et al., NCBI NLM NIH Bethesda N. Dak. 22894;        Altschul S. et al., above).

The known Smith-Waterman algorithm can also be used for determiningnucleotide identity.

Preferred parameters for the determination of “nucleotide identity” are,when using the BLASTN program (Altschul, S. et al., Journal of MolecularBiology 215 (1990), pages 403-410):

Expect Threshold: 10 Word size: 28 Match score: 1 Mismatch score: −2 Gapcosts: linear

The above parameters are the default parameters in nucleotide sequencecomparison.

The GAP program is also suitable for use with the above parameters.

An identity of 80% according to the above algorithm means, in connectionwith the present invention, 80% identity. The same applies to higheridentities.

The term “that are encoded by the intron-free nucleic acid sequences”makes clear that in a sequence comparison with the sequences given here,the nucleic acid sequences to be compared must be purified of anyintrons beforehand.

All stated percentages (%) are percentages by weight unless statedotherwise.

Methods of lowering enzymatic activities in microorganisms are known bya person skilled in the art.

In particular, techniques in molecular biology can be used for this. Aperson skilled in the art can find instructions on modification anddecrease of protein expression and the associated decrease in enzymeactivity especially for Candida tropicalis, in particular forinterrupting specified genes, in WO91/006660; WO03/100013; Picataggio etal. Mol Cell Biol. 1991 September; 11(9):4333-9; Rohrer et al. ApplMicrobiol Biotechnol. 1992 February; 36(5):650-4; Picataggio et al.Biotechnology (N Y). 1992 August; 10(8):894-8; Ueda et al. BiochimBiophys Acta. 2003 Mar. 17; 1631(2):160-8; Ko et al. Appl EnvironMicrobiol. 2006 June; 72(6):4207-13; Hara et al. Arch Microbiol. 2001November; 176(5):364-9; Kanayama et al. J Bacteriol. 1998 February;180(3): 690-8.

Cells preferred according to the invention are characterized in that thedecrease in enzymatic activity is achieved by modification of at leastone gene comprising one of the sequences selected from the previouslystated nucleic acid sequence groups A) and B), the modification beingselected from the group comprising, preferably consisting of, insertionof foreign DNA into the gene, deletion at least of parts of the gene,point mutations in the gene sequence and subjecting the gene to theinfluence of RNA interference or exchange of parts of the gene withforeign DNA, in particular of the promoter region.

Foreign DNA means, in this context, any DNA sequence that is “foreign”to the gene (and not to the organism), i.e. even Candida tropicalisendogenous DNA sequences can, in this context, function as “foreignDNA”.

In this context, it is in particular preferable for the gene to beinterrupted by insertion of a selection marker gene, therefore theforeign DNA is a selection marker gene, the insertion preferably havingbeen effected by homologous recombination into the gene locus.

In this context, it may be advantageous if the selection marker gene isexpanded with further functionalities, which in their turn makesubsequent removal from the gene possible, this can be achieved forexample with a Cre/loxP system, with Flippase Recognition Targets (FRT)or by homologous recombination.

Cells preferred according to the invention are characterized in thatthey are blocked in their β-oxidation at least partially, preferablycompletely, as this prevents outflow of substrate and therefore highertitres become possible.

Examples of Candida tropicalis cells partially blocked in theirβ-oxidation are described in EP0499622 as strains H41, H41B, H51, H45,H43, H53, H534, H534B and H435, from which a Candida tropicalis cellpreferred according to the invention is derived.

Other Candida tropicalis cells blocked for β-oxidation are described forexample in WO03/100013.

In this context, cells are preferred for which the β-oxidation is causedby an induced malfunction of at least one of the genes POX2, POX4 orPOX5.

Therefore, in this context, cells are preferred that are characterizedin that a Candida tropicalis cell preferred according to the inventionis derived from strains selected from the group comprising ATCC 20962and the Candida tropicalis HDC100 described in US2004/0014198.

The use of the cells according to the invention for the production ofω-hydroxycarboxylic acids or ω-hydroxycarboxylic acid esters alsocontributes to solution of the task facing the invention.

In particular, the use of the cells according to the invention for theproduction of ω-hydroxycarboxylic acids or ω-hydroxycarboxylic acidesters with a chain length of the carboxylic acid from 6 to 24,preferably 8 to 18 and especially preferably 10 to 16 carbon atoms,which are preferably linear, saturated and unsubstituted, and a chainlength of the alcohol component of the ester from 1 to 4, in particular1 or 2 carbon atoms, is advantageous. In this context, it is preferablefor the ω-hydroxycarboxylic acids to be 12-hydroxydodecanoic acid andfor the ω-hydroxycarboxylic acid ester to be 12-hydroxydodecanoic acidmethyl ester.

A preferred use is characterized according to the invention in thatpreferred cells according to the invention as described above are used.

Another contribution to solving the task facing the invention is made bya method of production of the C. tropicalis cell according to theinvention described above comprising the steps:

I) Preparation of a C. tropicalis cell, preferably a cell that isblocked in its β-oxidation at least partially, preferably completelyII) Modification of at least one gene comprising one of the intron-freenucleic acid sequences selected from the previously stated nucleic acidsequence groups A) and B) by insertion of foreign DNA, in particular ofDNA coding for a selection marker gene, into the gene, deletion of atleast parts of the gene, point mutations in the gene sequence andsubjecting the gene to the influence of RNA interference or exchange ofparts of the gene with foreign DNA, in particular of the promoterregion.

Another contribution to solving the task facing the invention is made bya method of production of ω-hydroxycarboxylic acids orω-hydroxycarboxylic acid esters, in particular of ω-hydroxycarboxylicacids or ω-hydroxycarboxylic acid esters with a chain length of thecarboxylic acid from 6 to 24, preferably 8 to 18 and especiallypreferably 10 to 16 carbon atoms, which are preferably linear, saturatedand unsubstituted, and a chain length of the alcohol component of theester from 1 to 4, in particular of 1 or 2 carbon atoms, in particularof 12-hydroxydodecanoic acid or 12-hydroxydodecanoic acid methyl estercomprising the steps

A) contacting a previously described cell according to the inventionwith a medium comprising a carboxylic acid or a carboxylic acid ester,in particular a carboxylic acid or a carboxylic acid ester with a chainlength of the carboxylic acid from 6 to 24, preferably 8 to 18 andespecially preferably 10 to 16 carbon atoms, which are preferablylinear, saturated and unsubstituted, and a chain length of the alcoholcomponent of the ester from 1 to 4 carbon atoms, in particulardodecanoic acid or dodecanoic acid methyl ester,B) cultivating the cell under conditions that enable the cell to formthe corresponding ω-hydroxycarboxylic acid or ω-hydroxycarboxylic acidesters from the carboxylic acid or the carboxylic acid ester andC) optionally isolating the ω-hydroxycarboxylic acid orω-hydroxycarboxylic acid esters that formed.

Preferred methods according to the invention use cells stated above asbeing preferred according to the invention.

Therefore, for example a method of production of 12-hydroxydodecanoicacid or 12-hydroxydodecanoic acid methyl ester comprising the steps

a) contacting a Candida tropicalis cell of the strain ATTC 20336 atleast partially blocked in its β-oxidation, which has, compared with itswild type, a reduced activity of at least one of the enzymes, which areencoded by the intron-free nucleic acid sequences selected from thepreviously stated nucleic acid sequence groups A) and B), the decreasein enzymatic activity being achieved by modification of a genecomprising one of the nucleic acid sequences selected from thepreviously stated nucleic acid sequence groups A) and B),wherein the modification consists of insertion of a selection markergene into the gene,with a medium comprising dodecanoic acid or dodecanoic acid methylester,b) cultivating the cell under conditions that enable the cell to formthe corresponding ω-hydroxycarboxylic acid or ω-hydroxycarboxylic acidesters from the carboxylic acid or the carboxylic acid ester andc) optionally isolating the ω-hydroxycarboxylic acid orω-hydroxycarboxylic acid esters that formedis quite especially preferred.

Suitable cultivation conditions for Candida tropicalis are known by aperson skilled in the art. In particular, suitable conditions for stepb) are those that are known by a person skilled in the art frombioconversion methods of production of dicarboxylic acids with Candidatropicalis.

These cultivation conditions are described for example in WO00/017380and WO00/015828.

Methods for isolating the ω-hydroxycarboxylic acid orω-hydroxycarboxylic acid esters that formed are known by a personskilled in the art. These are standard methods for isolating long-chaincarboxylic acids from aqueous solution, for example distillation orextraction, and can for example also be found in WO2009/077461.

It is advantageous to use the ω-hydroxycarboxylic acids orω-hydroxycarboxylic acid esters obtained by the method according to theinvention for the production of polymers, in particular polyesters.Moreover, lactones can also be produced from the ω-hydroxy carboxylicacids, and can then for example be used in their turn for the productionof polyesters.

Another advantageous use is to convert the ω-hydroxycarboxylic acids orω-hydroxycarboxylic acid esters to ω-aminocarboxylic acids orω-aminocarboxylic acid esters, in order to obtain polyamides aspolymers. The ω-aminocarboxylic acids or ω-aminocarboxylic acid esterscan also be converted first to the corresponding lactams, which can thenin their turn be converted using anionic, or also acid catalysis to apolyamide.

It is quite especially advantageous, in a first reaction step, toconvert the ω-hydroxycarboxylic acids or corresponding esters into theω-oxo-carboxylic acids or the corresponding esters and then to carry outamination of the oxo-group, e.g. in the course of reductive amination.

In this context, the use of 12-hydroxy dodecanoic acid or12-hydroxydodecanoic acid methyl ester for the production of polymers,in particular of polyamide 12, is especially preferred.

1. A mutant Candida tropicalis cell, which has, compared with a wildtype Candida tropicalis cell, a reduced activity of at least one ofenzyme that is encoded by an intron-free nucleic acid sequence selectedfrom groups A) and B) A) Seq ID No. 1, Seq ID No. 3, Seq ID No. 5, SeqID No. 7, Seq ID No. 9, Seq ID No. 11, Seq ID No. 13, Seq ID No. 15, SeqID No. 17, Seq ID No. 19, Seq ID No. 21, Seq ID No. 23, Seq ID No. 25,Seq ID No. 27, Seq ID No. 29, Seq ID No. 31, Seq ID No. 33, Seq ID No.35, Seq ID No. 37, Seq ID No. 39, Seq ID No. 41, Seq ID No. 43, Seq IDNo. 45, Seq ID No. 47, Seq ID No. 49, Seq ID No. 51, Seq ID No. 53, SeqID No. 55, Seq ID No. 57, Seq ID No. 59, Seq ID No. 61, Seq ID No. 63,Seq ID No. 65 and Seq ID No. 67 B) a sequence that is 80% identical toat least to one sequence selected from the group consisting of Seq IDNo. 1, Seq ID No. 3, Seq ID No. 5, Seq ID No. 7, Seq ID No. 9, Seq IDNo. 11, Seq ID No. 13, Seq ID No. 15, Seq ID No. 17, Seq ID No. 19, SeqID No. 21, Seq ID No. 23, Seq ID No. 25, Seq ID No. 27, Seq ID No. 29,Seq ID No. 31, Seq ID No. 33, Seq ID No. 35, Seq ID No. 37, Seq ID No.39, Seq ID No. 41, Seq ID No. 43, Seq ID No. 45, Seq ID No. 47, Seq IDNo. 49, Seq ID No. 51, Seq ID No. 53, Seq ID No. 55, Seq ID No. 57, SeqID No. 59, Seq ID No. 61, Seq ID No. 63, Seq ID No. 65 and Seq ID No.67.
 2. The Candida tropicalis cell according to claim 1, wherein thedecrease in enzymatic activity is achieved by modification of thenucleic acid sequence in the cell, wherein the modification is selectedfrom the group consisting of insertion of foreign DNA into the nucleicacid sequence in the cell, deletion of at least parts of the nucleicacid sequence in the cell, a point mutation in the nucleic acid sequencein the cell, subjecting the nucleic acid sequence in the cell to RNAinterference and exchanging a part of the nucleic acid sequence in thecell with foreign DNA.
 3. The Candida tropicalis cell according to claim2, wherein the foreign DNA is a selection marker gene.
 4. The Candidatropicalis cell according to claim 1, wherein the cell is blocked atleast partially in its β-oxidation.
 5. The Candida tropicalis cellaccording to claim 1, which is derived from a strain selected from thegroup consisting of Candida tropicalis H41, Candida tropicalis H41B,Candida tropicalis H51, Candida tropicalis H45, Candida tropicalis H43,Candida tropicalis H53, Candida tropicalis H534, Candida tropicalis534B, Candida tropicalis H435, Candida tropicalis ATCC20962 and Candidatropicalis HDC100.
 6. The Candida tropicalis cell according to claim 5,which is derived from Candida tropicalis ATCC20962 or Candida tropicalisHDC100.
 7. A method for producing ω-hydroxycarboxylic acid orω-hydroxycarboxylic acid ester, the method comprising a) contacting theCandida tropicalis cell according to claim 1 with a medium comprising acarboxylic acid or a carboxylic acid ester, b) cultivating the cellunder conditions to form the corresponding ω-hydroxycarboxylic acid orω-hydroxycarboxylic acid esters from the carboxylic acid or thecarboxylic acid ester and c) optionally isolating theω-hydroxycarboxylic acid or ω-hydroxycarboxylic acid esters that formed.8. A method of producing a C. tropicalis cell according to claim 1, themethod comprising: I) preparing a C. tropicalis cell and II) modifyingat least one gene comprising one of the sequences selected from thenucleic acid sequence groups A) and B) stated in claim 1 by insertion offoreign DNA into the gene, deletion at least of a part of the gene, apoint mutation in the gene sequence, subjecting the gene to RNAinterference and exchanging a part of the gene with foreign DNA.
 9. Themethod according to claim 7, wherein the ω-hydroxycarboxylic acid orω-hydroxycarboxylic acid ester is a ω-hydroxycarboxylic acid orω-hydroxycarboxylic acid ester with a chain length of the carboxylicacid from 6 to 24 carbon atoms and a chain length of the alcoholcomponent of the ester from 1 to 4 carbon atoms
 10. The method accordingto claim 7, wherein the ω-hydroxycarboxylic acid or ω-hydroxycarboxylicacid ester is a 12-hydroxydodecanoic acid or 12-hydroxydodecanoic acidmethyl ester.
 11. A method according to claim 7, wherein the Candidatropicalis cells are derived from a strain selected from the groupconsisting of Candida tropicalis H41, Candida tropicalis H41B, Candidatropicalis H51, Candida tropicalis H45, Candida tropicalis H43, Candidatropicalis H53, Candida tropicalis H534, Candida tropicalis 534B,Candida tropicalis H435, Candida tropicalis ATCC20962 and Candidatropicalis HDC100 and wherein the cells are at least partially blockedin their β-oxidation.
 12. A method of manufacturing a polymer, themethod comprising polymerizing the ω-hydroxycarboxylic acid or of theω-hydroxycarboxylic acid ester obtained by the method according to claim7.